Since 1996, there have been numerous reports of human infections with avian influenza A viruses of subtypes H5N1, H7N7 and H9N2. In 2013, a new avian influenza virus strain of H7N9 subtype emerged in China causing more than 450 infections in humans resulting in 175 deaths so far. Although human-to-human transmission has been infrequent and limited, genetic reassortment between avian and human/porcine influenza viruses or mutations in some of the genes leading to virus replication in the upper respiratory tract in humans could result in the generation of a novel pandemic influenza virus strain that not only would infect but also effectively transmit among the human population which would have little or no immunity to the new virus. Our immunogenicity and protective efficacy studies showed that adenovirus (Ad) vector-based H5N1, H7N7 and H9N2 vaccines provide excellent humoral and cell-mediated immune responses leading to complete protection against challenges with H5N1, H7N7 and H9N2 influenza viruses. To further improve the breadth of protective efficacy, inclusion of neuraminidase (NA), in addition to hemagglutinin (HA) and nucleoprotein (NP), in the Ad vector-based vaccine formulations will be explored (first generation vaccines). The other strategy to enhance the breadth of protective efficacy against avian influenza viruses will be to include relatively conserved domains of influenza proteins for multi-epitope-based vaccines (second generation vaccines). This proposal is based on the hypothesis that a combination of heterosubtypic cell-mediated immunity against NP and the cross-reactive (not necessarily cross-neutralizing) humoral immune responses against other immunogenic proteins or domains will provide broad protection against potential pandemic H5N1, H7N9 or H9N2 avian influenza viruses as well as other H5, H7 or H9 divergent viruses. The vaccine approach will be based on the bovine adenovirus vector which induces improved levels of protective immune responses even in the presence of high levels of neutralizing antibodies against human Ad. The goals of this revised competitive renewal proposal are: Aim 1) Development and evaluation of the immunogenicity and protective efficacy of broadly protective Ad-based influenza vaccines (HA, NP & NA-based or immunogenic multi-epitope-based) against H5N1, H7N9, and H9N2 avian influenza viruses in mice; Aim 2) Evaluation of the immunogenicity and protective efficacy of selected broadly protective Ad-based vaccines against H5N1, H7N9, and H9N2 viruses in ferrets; and Aim 3) Determination of the longevity of protective influenza immunity and the decline of Ad vector immunity with time and its effect on repeat immunization with the same Ad vector. Our efforts will be directed towards the generation of broadly protective vaccines against H5, H7 and H9 influenza subtypes which would significantly lower morbidity, hinder transmission and prevent mortality in a pandemic situation before a strain-matched vaccine can be produced. The outcome of this research will significantly impact the development efforts for better vaccines against seasonal influenza viruses.